SUMMARY Neovessel guidance in angiogenesis Vascular connectivity between adjacent vessel beds within and between tissue compartments is an essential aspect of any successful neovascularization process. To establish new connections, growing neovessels must locate other vascular elements during angiogenesis, often crossing matrix and other tissue-associated boundaries and interfaces. This is perhaps best highlighted in situations of tissue grafting (e.g. tissue free flaps) during which the vasculature of the graft must cross the graft-host tissue interface before connecting to the surrounding host circulation in order for the graft to survive. An inability for a tissue graft vasculature to connect to the host vasculature is the primary cause for tissue graft failure and necrosis. How growing neovessels traverse any tissue interface, whether part of the native tissue structure or secondary to a grafting procedure, is not known. In a series of preliminary experiments, we have determined that actively growing neovessels are unable to spontaneously cross a stroma-stroma interface during angiogenesis. Our published findings that tissue stromal biomechanics, specifically the biophysical aspects of the matrix, has a profound influence on the direction and branching of growing neovessels suggests that this interface is biomechanically incompatible with interface invasion. In addition, we have evidence that a sub-population of tissue-resident, stromal macrophages facilitates neovessel invasion of the stromal interface during angiogenesis through a VEGF-A-dependent process. Importantly, it appears that stromal cells need to migrate across the interface in order to promote interface invasion by the neovessels, suggesting that spatial gradients of VEGF-A are required. Based on these observations, we hypothesize that the graded angiogenic factor signals overcome the biomechanical barriers to directed angiogenesis caused by a stroma-stroma interface to promote interface neovascular invasion. The project will involve a combination of in vitro and computational models of angiogenesis across tissue-tissue boundaries to test this hypothesis and determine the mechanism by which stromal cells use VEGF-A signaling to regulate neovessel behavior in coordination with stroma biomechanical dynamics. These studies will provide new insights into a poorly understood aspect of vascular biology and tissue-vascular dynamics as well as create opportunities for therapeutic strategies to facilitate tissue healing, improving angiogenesis-based treatments, and tissue grafting/transplantation.